Flt3 receptor antagonists for the treatment or the prevention of pain disorders

ABSTRACT

The present invention relates to FLT3 receptor antagonists or inhibitors of FLT3 receptor gene expression for the treatment or the prevention of pain disorders.

FIELD OF THE INVENTION

The present invention relates to FLT3 receptor antagonists or inhibitorsof FLT3 receptor gene expression for the treatment or the prevention ofpain disorders.

BACKGROUND OF THE INVENTION

Somatic sensations such as warming, cooling, gentle touch and pain areeach initiated by activation of sensory neurons. Specific types ofsensory neurons, whose cell bodies are located in dorsal root andtrigeminal ganglia, subserve different sensory modalities. Specializedsensory neurons called nociceptors are responsible for the transductionof painful thermal and mechanical stimulation of the skin. Knowledgeabout molecules and ion channels that are necessary for the normaltransduction of painful thermal and mechanical stimuli is stillincomplete. It has been postulated that thermosensitive ion channels ofthe TRP family are important for the transduction of noxious heat orcold by nociceptive sensory neurons (Jordt et al., 2003). The mostcomplete evidence exists for the capsaicin activated ion channel TRPV1that can be activated by thermal stimuli in the noxious range. Micelacking TRPV1 have altered pain behavior and do not respond to thenoxious irritant capsaicin. An important feature of pain is the factthat injury and inflammation leads to heightened sensitivity to stimulithat would normally be only mildly painful. This phenomenon is calledhyperalgesia, and the prevention of hyperalgesia is a hallmark ofeffective analgesia. TRPV1 may become an important analgesic targetbecause this channel is required for the expression of thermalhyperalgesia provoked by inflammation (Caterina et al., 2000; Davis etal., 2000).

Moreover, molecules up-regulated in inflamed tissue such as nerve growthfactor (NGF) can sensitize peripheral nociceptors to thermal stimuli.NGF signaling via its receptor tyrosine kinase TrkA constitutes aphysiological mediator of inflammatory hyperalgesia. It has been knownfor many years that the dorsal root ganglion (DRG) neurons that requireNGF are all nociceptors. NGF can produce a profound and long lastingthermal and mechanical hyperalgesia in man and animals. NGF can alsopotentiate TRPV1 mediated and noxious heat activated ionic currents inisolated DRG neurons. Indeed, NGF injected into animals produces thermalhyperalgesia that requires the presence of TRPV1 (Chuang et al., 2001).

Around half of the nociceptors in the adult DRG possess TrkA receptors;the remainder, defined by the expression of c-Ret, downregulate TrkAduring early postnatal development. The receptor tyrosine kinase c-Retmediates signals elicited by the glial-derived neurotophic factor (GDNF)ligand family. The c-Ret receptor and its co-receptors GFRα2 and 3 arepresent in nociceptive neurons, some of which are heat sensitive andexpress TRPV1 receptors. Indeed, there is some evidence for a role ofthe GDNF family ligands neurturin and artemin in regulating noxious heattransduction by sensory neurons (Malin et al., 2006).

In addition to the Trk and c-Ret receptors, sensory neurons are known toexpress other receptor tyrosine kinases like c-Kit, the receptor forstem cell factor (SCF). Thus, the European patent application No EP 2068 152 discloses that the central role for SCF and its receptor, c-Kit,in tuning the responsiveness of sensory neurons to natural stimuli andthat c-Kit can now be grouped with a small family of receptor tyrosinekinases, including c-Ret and TrkA, that control the transductionproperties of sensory neurons. Said patent application claims the use ofa c-kit receptor antagonist such as the small molecule drug imatinib fortreating or preventing a disorder selected from pain, hyperalgesia andinflammatory pain.

However, no investigation on FLT3 receptor pathway (another tyrosinekinase receptor) has been made until now on pain regulation.

SUMMARY OF THE INVENTION

The present invention relates to a FLT3 receptor antagonist for thetreatment or the prevention of pain disorders.

The present invention also relates to an inhibitor of FLT3 receptor geneexpression for the treatment or the prevention of pain disorders.

DETAILED DESCRIPTION OF THE INVENTION Definitions

Throughout the specification, several terms are employed and are definedin the following paragraphs.

The terms “FLT3” or “FLT3 receptor” (fins-related tyrosine kinase 3),also known as the CD135, Ly72, Flk-2, Flt-3 or B230315G04, are usedinterchangeably and have their general meaning in the art. The FLT3receptor can be from any source, but typically is a mammalian (e.g.,human and non-human primate) FLT3 receptor, particularly a human FLT3receptor.

The terms “FL” or “FLT3-Ligand” are used interchangeably and have theirgeneral meaning in the art. They refer to the cytokine which is anatural ligand of the FLT3 receptor. FL can be from any source, buttypically is a mammalian (e.g., human and non-human primate) FL,particularly a human FL.

An “inhibitor of gene expression” refers to a natural or syntheticcompound that has a biological effect to inhibit or significantly reducethe expression of a gene. Consequently an “inhibitor of FLT3 receptorgene expression” refers to a natural or synthetic compound that has abiological effect to inhibit or significantly reduce the expression ofthe gene encoding for the FLT3 receptor.

By “receptor antagonist” is meant a natural or synthetic compound thathas a biological effect opposite to that of a receptor agonist. The termis used indifferently to denote a “true” antagonist and an inverseagonist of a receptor. A “true” receptor antagonist is a compound whichbinds the receptor and blocks the biological activation of the receptor,and thereby the action of the receptor agonist, for example, bycompeting with the agonist for said receptor. An inverse agonist is acompound which binds to the same receptor as the agonist but exerts theopposite effect. Inverse agonists have the ability to decrease theconstitutive level of receptor activation in the absence of an agonist.

The terms “FLT3 receptor antagonist” includes any entity that, uponadministration to a patient, results in inhibition or down-regulation ofa biological activity associated with activation of the FLT3 receptor byFL in the patient, including any of the downstream biological effectsotherwise resulting from the binding to FLT3 receptor with FL. Such FLT3receptor antagonists include any agent that can block FLT3 receptoractivation or any of the downstream biological effects of FLT3 receptoractivation. For example, such a FLT3 receptor antagonist (e.g. a smallorganic molecule, an antibody directed against FLT3) can act byoccupying the ligand binding site or a portion thereof of the FLT3receptor, thereby making FLT3 receptor inaccessible to its naturalligand, FL, so that its normal biological activity is prevented orreduced. The term FLT3 receptor antagonist includes also any agent ableto interact with the natural ligand of FLT3, namely FL. Said agent maybe an antibody directed against FL which can block the interactionbetween FL and FLT3 or which can block the activity of FL (“neutralizingantibody”).

The term “blocking the interaction”, “inhibiting the interaction” or“inhibitor of the interaction” are used herein to mean preventing orreducing the direct or indirect association of one or more molecules,peptides, proteins, enzymes or receptors; or preventing or reducing thenormal activity of one or more molecules, peptides, proteins, enzymes,or receptors. The term “inhibitor of the interaction between FLT3 andFL” is a molecule which can prevent the interaction between FLT3 and FLby competition or by fixing to one of the molecule.

In the context of the present invention, when FLT3 receptor antagonistsare small organic molecules, said antagonists are preferably selectivefor the FLT3 receptor as compared with the other tyrosine kinasereceptors, such as c-Kit receptor. By “selective” it is meant that theaffinity of the antagonist for the FLT3 receptor is at least 10-fold,preferably 25-fold, more preferably 100-fold, still preferably 150-foldhigher than the affinity for the other tyrosine kinase receptors (c-Kitreceptor). Selectivity of a FLT3 receptor antagonists may be assayed forinstance by carrying out biochemical kinase binding assays such asKinomeScan kinase binding assays as described in Fabian et al. 2005 andKaraman et al. 2008. For the FLT3 assay, a kinase construct that spannedthe catalytic domain only (amino acids 592 to 969 in NP 004110.2) may beused. This construct does not include the juxtamembrane domain and isdesigned to measure the intrinsic binding affinity of the open FLT3active site for inhibitors as previously described in Zarrinkar et al.2009.

The affinity of an antagonist for FLT3 receptor may be quantified bymeasuring the activity of FLT3 receptor in the presence a range ofconcentrations of said antagonist in order to establish a dose-responsecurve. From that dose response curve, an IC₅₀ value may be deduced whichrepresents the concentration of antagonist necessary to inhibit 50% ofthe response to an agonist in defined concentration. The IC₅₀ value maybe readily determined by the one skilled in the art by fitting thedose-response plots with a dose-response equation as described by DeLean et al. 1979. IC₅₀ values can be converted into affinity constant(Ki) using the assumptions of Cheng and Prusoff (1973).

The term “small organic molecule” refers to a molecule of a sizecomparable to those organic molecules generally used in pharmaceuticals.The term excludes biological macromolecules (e. g., proteins, nucleicacids, etc.). Preferred small organic molecules range in size up toabout 5000 Da, more preferably up to 2000 Da, and most preferably up toabout 1000 Da.

As used herein, the term “subject” denotes a mammal, such as a rodent, afeline, a canine, and a primate. Preferably, a subject according to theinvention is a human.

The term “pain disorders” refers to disorder selected in the groupconsisting of acute pain, chronic pain, neuropathic pain, inflammatorypain, low back pain, post-operative pain, cancer pain, vascular headachesuch as migraine, fibromyalgia, hyperalgesia such as mechanical andthermal hyperalgesia, allodynia such as thermal and mechanicalallodynia, peripheral sensitization of pain mechanisms and centralsensitization of pain mechanisms.

In its broadest meaning, the term “treating” or “treatment” refers toreversing, alleviating, inhibiting the progress of, or preventing thedisorder or condition to which such term applies, or one or moresymptoms of such disorder or condition.

In particular, “prevention” of pain may refer to the administration ofthe compounds of the present invention prevent the symptoms of pain.

“Pharmaceutically” or “pharmaceutically acceptable” refers to molecularentities and compositions that do not produce an adverse, allergic orother untoward reaction when administered to a mammal, especially ahuman, as appropriate. A pharmaceutically acceptable carrier orexcipient refers to a non-toxic solid, semi-solid or liquid filler,diluent, encapsulating material or formulation auxiliary of any type.

Therapeutic Methods and Uses

The present invention provides methods and compositions (such aspharmaceutical compositions) for treating or preventing pain disorders.

According to a first aspect, the invention relates to a FLT3 receptorantagonist for treating or preventing pain disorders.

In one embodiment, the FLT3 receptor antagonist may be a low molecularweight antagonist, e. g. a small organic molecule.

Exemplary FLT3 receptor antagonists that are contemplated by theinvention include but are not limited to those described in Sternberg etal. 2004 and in International Patent Application Nos WO 2002032861, WO2002092599, WO 2003035009, WO 2003024931, WO 2003037347, WO 2003057690,WO 2003099771, WO 2004005281, WO 2004016597, WO 2004018419, WO2004039782, WO 2004043389, WO 2004046120, WO 2004058749, WO 2004058749,WO 2003024969, WO 2006/138155, WO 2007/048088 and WO 2009/095399 whichare incorporated herein by reference.

More particularly, FLT3 receptor antagonists may consist in FLT3 kinaseinhibitors. Exemplary of FLT3 kinase inhibitors that are contemplatedinclude AG1295 and AG1296; Lestaurtinib (also known as CEP-701, formerlyKT-5555, Kyowa Hakko, licensed to Cephalon); CEP-5214 and CEP-7055(Cephalon); CHIR-258 (Chiron Corp.); GTP 14564 (Merk Biosciences UK).Midostaurin (also known as PKC 412 Novartis AG); MLN-608 (MillenniumUSA); MLN-518 (formerly CT53518, COR Therapeutics Inc., licensed toMillennium Pharmaceuticals Inc.); MLN-608 (Millennium PharmaceuticalsInc.); SU-11248 (Pfizer USA); SU-11657 (Pfizer USA); SU-5416 andSU-5614; THRX-165724 (Theravance Inc.); AMI-10706 (Theravance Inc.);VX-528 and VX-680 (Vertex Pharmaceuticals USA, licensed to Novartis(Switzerland), Merck & Co USA); and XL 999 (Exelixis USA).

In a preferred embodiment, the FLT3 receptor antagonist is a selectiveFLT3 receptor antagonist.

Exemplary selective FLT3 receptor antagonists that are contemplated bythe invention include but are not limited to those described inZarrinkar et al. 2009 and in International Patent Applications No WO2007/109120 and WO 2009/061446 which are incorporated herein byreference. Accordingly, in a most preferred embodiment, the selectiveFLT3 receptor antagonist is the compound known as AC220 orN-(5-tert-butyl-isoxazol-3-yl)-N′-{4-[7-(2-morpholin-4-yl-ethoxy)imidazo[2,1-b][1,3]benzothiazol-2-yl]phenyl}ureadihydrochloride. Said AC220 compound may be made by methods known in theart, for example, as described in the international patent applicationWO 2007/109120.

In another embodiment, the FLT3 receptor antagonist is an inhibitor ofthe interaction between FLT3 and FL.

The compounds that inhibit the interaction between FLT3 and FL encompassthose compounds that bind either to the FLT3 or to FL, provided that thebinding of the said compounds of interest then prevent the interactionbetween FLT3 and FL. Accordingly, said compounds may be selected fromthe group consisting of peptides, peptidomimetics, small organicmolecules, antibodies, aptamers or nucleic acids.

In another embodiment, the FLT3 receptor antagonist may consist in anantibody (the term including antibody fragment) that can block FLT3receptor activation. In particular, the FLT3 receptor antagonist mayconsist in an antibody directed against the FLT3 receptor or FL, in sucha way that said antibody impairs the binding of a FL to FLT3.

In a particular embodiment, the FLT3 receptor antagonist may be ananti-FLT3 neutralizing antibody such as IMC-EB10 described in Li et al.,2004 and in US patent application No US 2009/0297529 which areincorporated herein by reference.

In another particular embodiment, the FLT3 receptor antagonist may be ananti-FL neutralizing antibody.

Antibodies directed against the FLT3 receptor or FL can be raisedaccording to known methods by administering the appropriate antigen orepitope to a host animal selected, e.g., from pigs, cows, horses,rabbits, goats, sheep, and mice, among others. Various adjuvants knownin the art can be used to enhance antibody production. Althoughantibodies useful in practicing the invention can be polyclonal,monoclonal antibodies are preferred. Monoclonal antibodies against FLT3receptor or FL can be prepared and isolated using any technique thatprovides for the production of antibody molecules by continuous celllines in culture. Techniques for production and isolation include butare not limited to the hybridoma technique originally described byKohler and Milstein (1975); the human B-cell hybridoma technique (Coteet al., 1983); and the EBV-hybridoma technique (Cole et al. 1985).Alternatively, techniques described for the production of single chainantibodies (see, e.g., U.S. Pat. No. 4,946,778) can be adapted toproduce anti-FLT3, or anti-FL single chain antibodies. FLT3 receptorantagonists useful in practicing the present invention also includeanti-FLT3, or anti-FL antibody fragments including but not limited toF(ab′)2 fragments, which can be generated by pepsin digestion of anintact antibody molecule, and Fab fragments, which can be generated byreducing the disulfide bridges of the F(ab′)2 fragments. Alternatively,Fab and/or scFv expression libraries can be constructed to allow rapididentification of fragments having the desired specificity to FLT3receptor.

Humanized anti-FLT3 receptor or anti-FL antibodies and antibodyfragments therefrom can also be prepared according to known techniques.“Humanized antibodies” are forms of non-human (e.g., rodent) chimericantibodies that contain minimal sequence derived from non-humanimmunoglobulin. For the most part, humanized antibodies are humanimmunoglobulins (recipient antibody) in which residues from ahypervariable region (CDRs) of the recipient are replaced by residuesfrom a hypervariable region of a non-human species (donor antibody) suchas mouse, rat, rabbit or nonhuman primate having the desiredspecificity, affinity and capacity. In some instances, framework region(FR) residues of the human immunoglobulin are replaced by correspondingnon-human residues. Furthermore, humanized antibodies may compriseresidues that are not found in the recipient antibody or in the donorantibody. These modifications are made to further refine antibodyperformance. In general, the humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the hypervariable loops correspondto those of a non-human immunoglobulin and all or substantially all ofthe FRs are those of a human immunoglobulin sequence. The humanizedantibody optionally also will comprise at least a portion of animmunoglobulin constant region (Fc), typically that of a humanimmunoglobulin. Methods for making humanized antibodies are described,for example, by Winter (U.S. Pat. No. 5,225,539) and Boss (Celltech,U.S. Pat. No. 4,816,397).

Then after raising antibodies directed against the FLT3 receptor or FLas above described, the skilled man in the art can easily select thoseblocking FLT3 receptor activation.

In another embodiment the FLT3 receptor antagonist is an aptamerdirected against FLT3 or FL. Aptamers are a class of molecule thatrepresents an alternative to antibodies in term of molecularrecognition. Aptamers are oligonucleotide or oligopeptide sequences withthe capacity to recognize virtually any class of target molecules withhigh affinity and specificity. Such ligands may be isolated throughSystematic Evolution of Ligands by EXponential enrichment (SELEX) of arandom sequence library, as described in Tuerk C. and Gold L., 1990. Therandom sequence library is obtainable by combinatorial chemicalsynthesis of DNA. In this library, each member is a linear oligomer,eventually chemically modified, of a unique sequence. Possiblemodifications, uses and advantages of this class of molecules have beenreviewed in Jayasena S. D., 1999. Peptide aptamers consists of aconformationally constrained antibody variable region displayed by aplatform protein, such as E. coli Thioredoxin A that are selected fromcombinatorial libraries by two hybrid methods (Colas et al., 1996).

Then after raising aptamers directed against the FLT3 receptor or FL asabove described, the skilled man in the art can easily select thoseblocking FLT3 receptor activation.

Another aspect of the invention relates to an inhibitor of FLT3 receptorgene expression for treating or preventing pain disorders.

Inhibitors of FLT3 receptor gene expression for use in the presentinvention may be based on anti-sense oligonucleotide constructs.Anti-sense oligonucleotides, including anti-sense RNA molecules andanti-sense DNA molecules, would act to directly block the translation ofFLT3 receptor mRNA by binding thereto and thus preventing proteintranslation or increasing mRNA degradation, thus decreasing the level ofFLT3 receptors, and thus activity, in a cell. For example, antisenseoligonucleotides of at least about 15 bases and complementary to uniqueregions of the mRNA transcript sequence encoding FLT3 receptor can besynthesized, e.g., by conventional phosphodiester techniques andadministered by e.g., intravenous injection or infusion. Methods forusing antisense techniques for specifically inhibiting gene expressionof genes whose sequence is known are well known in the art (e.g. seeU.S. Pat. Nos. 6,566,135; 6,566,131; 6,365,354; 6,410,323; 6,107,091;6,046,321; and 5,981,732).

Small inhibitory RNAs (siRNAs) can also function as inhibitors of FLT3receptor gene expression for use in the present invention. FLT3 receptorgene expression can be reduced by contacting a subject or cell with asmall double stranded RNA (dsRNA), or a vector or construct causing theproduction of a small double stranded RNA, such that FLT3 receptor geneexpression is specifically inhibited (i.e. RNA interference or RNAi).Methods for selecting an appropriate dsRNA or dsRNA-encoding vector arewell known in the art for genes whose sequence is known (e.g. seeTuschl, T. et al. (1999); Elbashir, S. M. et al. (2001); Hannon, G J.(2002); McManus, M T. et al. (2002); Brummelkamp, T R. et al. (2002);U.S. Pat. Nos. 6,573,099 and 6,506,559; and International PatentPublication Nos. WO 01/36646, WO 99/32619, and WO 01/68836).

In one embodiment, the sequence of the siRNA targeting FLT3 isrepresented by SEQ ID NO: 1.

In another embodiment, the sequence of the siRNA targeting FLT3 isrepresented by SEQ ID NO: 2.

In another embodiment, the sequence of the siRNA targeting FLT3 isrepresented by SEQ ID NO: 3.

In still another embodiment, the sequence of the siRNA targeting FLT3 isrepresented by SEQ ID NO: 4.

In a preferred embodiment, a pool of siRNAs targeting FLT3 may be used.Such pool may comprise at least 2 siRNAs selected from SEQ ID NO: 1, SEQID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4.

It must be further noted that such a pool of siRNAs targeting FLT3 maybe used as inhibitors of murine and human FLT3 receptor gene expression.

Ribozymes can also function as inhibitors of FLT3 receptor geneexpression for use in the present invention. Ribozymes are enzymatic RNAmolecules capable of catalyzing the specific cleavage of RNA. Themechanism of ribozyme action involves sequence specific hybridization ofthe ribozyme molecule to complementary target RNA, followed byendonucleolytic cleavage. Engineered hairpin or hammerhead motifribozyme molecules that specifically and efficiently catalyzeendonucleolytic cleavage of FLT3 receptor mRNA sequences are therebyuseful within the scope of the present invention. Specific ribozymecleavage sites within any potential RNA target are initially identifiedby scanning the target molecule for ribozyme cleavage sites, whichtypically include the following sequences, GUA, GUU, and GUC. Onceidentified, short RNA sequences of between about 15 and 20ribonucleotides corresponding to the region of the target genecontaining the cleavage site can be evaluated for predicted structuralfeatures, such as secondary structure, that can render theoligonucleotide sequence unsuitable. The suitability of candidatetargets can also be evaluated by testing their accessibility tohybridization with complementary oligonucleotides, using, e.g.,ribonuclease protection assays.

Both antisense oligonucleotides and ribozymes useful as inhibitors ofFLT3 receptor gene expression can be prepared by known methods. Theseinclude techniques for chemical synthesis such as, e.g., by solid phasephosphoramadite chemical synthesis. Alternatively, anti-sense RNAmolecules can be generated by in vitro or in vivo transcription of DNAsequences encoding the RNA molecule. Such DNA sequences can beincorporated into a wide variety of vectors that incorporate suitableRNA polymerase promoters such as the T7 or SP6 polymerase promoters.Various modifications to the oligonucleotides of the invention can beintroduced as a means of increasing intracellular stability andhalf-life. Possible modifications include but are not limited to theaddition of flanking sequences of ribonucleotides ordeoxyribonucleotides to the 5′ and/or 3′ ends of the molecule, or theuse of phosphorothioate or 2′-O-methyl rather than phosphodiesteraselinkages within the oligonucleotide backbone.

Antisense oligonucleotides siRNAs and ribozymes of the invention may bedelivered in vivo alone or in association with a vector. In its broadestsense, a “vector” is any vehicle capable of facilitating the transfer ofthe antisense oligonucleotide siRNA or ribozyme nucleic acid to thecells and preferably cells expressing FLT3 receptor. Preferably, thevector transports the nucleic acid to cells with reduced degradationrelative to the extent of degradation that would result in the absenceof the vector. In general, the vectors useful in the invention include,but are not limited to, plasmids, phagemids, viruses, other vehiclesderived from viral or bacterial sources that have been manipulated bythe insertion or incorporation of the antisense oligonucleotide siRNA orribozyme nucleic acid sequences. Viral vectors are a preferred type ofvector and include, but are not limited to nucleic acid sequences fromthe following viruses: retrovirus, such as moloney murine leukemiavirus, harvey murine sarcoma virus, murine mammary tumor virus, androuse sarcoma virus; adenovirus, adeno-associated virus; SV40-typeviruses; polyoma viruses; Epstein-Barr viruses; papilloma viruses;herpes virus; vaccinia virus; polio virus; and RNA virus such as aretrovirus. One can readily employ other vectors not named but known tothe art.

Preferred viral vectors are based on non-cytopathic eukaryotic virusesin which non-essential genes have been replaced with the gene ofinterest. Non-cytopathic viruses include retroviruses (e.g.,lentivirus), the life cycle of which involves reverse transcription ofgenomic viral RNA into DNA with subsequent proviral integration intohost cellular DNA. Retroviruses have been approved for human genetherapy trials. Most useful are those retroviruses that arereplication-deficient (i.e., capable of directing synthesis of thedesired proteins, but incapable of manufacturing an infectiousparticle). Such genetically altered retroviral expression vectors havegeneral utility for the high-efficiency transduction of genes in vivo.Standard protocols for producing replication-deficient retroviruses(including the steps of incorporation of exogenous genetic material intoa plasmid, transfection of a packaging cell lined with plasmid,production of recombinant retroviruses by the packaging cell line,collection of viral particles from tissue culture media, and infectionof the target cells with viral particles) are provided in Kriegler, 1990and in Murry, 1991).

Preferred viruses for certain applications are the adeno-viruses andadeno-associated viruses, which are double-stranded DNA viruses thathave already been approved for human use in gene therapy. Theadeno-associated virus can be engineered to be replication deficient andis capable of infecting a wide range of cell types and species. Itfurther has advantages such as, heat and lipid solvent stability; hightransduction frequencies in cells of diverse lineages, includinghemopoietic cells; and lack of superinfection inhibition thus allowingmultiple series of transductions. Reportedly, the adeno-associated viruscan integrate into human cellular DNA in a site-specific manner, therebyminimizing the possibility of insertional mutagenesis and variability ofinserted gene expression characteristic of retroviral infection. Inaddition, wild-type adeno-associated virus infections have been followedin tissue culture for greater than 100 passages in the absence ofselective pressure, implying that the adeno-associated virus genomicintegration is a relatively stable event. The adeno-associated virus canalso function in an extrachromosomal fashion.

Other vectors include plasmid vectors. Plasmid vectors have beenextensively described in the art and are well known to those of skill inthe art. See e.g. Sambrook et al., 1989. In the last few years, plasmidvectors have been used as DNA vaccines for delivering antigen-encodinggenes to cells in vivo. They are particularly advantageous for thisbecause they do not have the same safety concerns as with many of theviral vectors. These plasmids, however, having a promoter compatiblewith the host cell, can express a peptide from a gene operativelyencoded within the plasmid. Some commonly used plasmids include pBR322,pUC18, pUC19, pRC/CMV, SV40, and pBlueScript. Other plasmids are wellknown to those of ordinary skill in the art. Additionally, plasmids maybe custom designed using restriction enzymes and ligation reactions toremove and add specific fragments of DNA. Plasmids may be delivered by avariety of parenteral, mucosal and topical routes. For example, the DNAplasmid can be injected by intramuscular, intradermal, subcutaneous, orother routes. It may also be administered by intranasal sprays or drops,rectal suppository and orally. It may also be administered into theepidermis or a mucosal surface using a gene-gun. The plasmids may begiven in an aqueous solution, dried onto gold particles or inassociation with another DNA delivery system including but not limitedto liposomes, dendrimers, cochleate and microencapsulation.

Another aspect of the invention relates to a method for treating orpreventing pain disorders comprising administering a subject in needthereof with a FLT3 receptor antagonist or an inhibitor of FLT3 receptorgene expression.

FLT3 receptor antagonists or inhibitors of FLT3 receptor gene expressionmay be administered in the form of a pharmaceutical composition, asdefined below. Preferably, said antagonist or inhibitor is administeredin a therapeutically effective amount.

By a “therapeutically effective amount” is meant a sufficient amount ofthe FLT3 receptor antagonist or inhibitor of FLT3 receptor geneexpression to treat or to prevent insulin resistance at a reasonablebenefit/risk ratio applicable to any medical treatment.

It will be understood that the total daily usage of the compounds andcompositions of the present invention will be decided by the attendingphysician within the scope of sound medical judgment. The specifictherapeutically effective dose level for any particular patient willdepend upon a variety of factors including the disorder being treatedand the severity of the disorder; activity of the specific compoundemployed; the specific composition employed, the age, body weight,general health, sex and diet of the patient; the time of administration,route of administration, and rate of excretion of the specific compoundemployed; the duration of the treatment; drugs used in combination orcoincidential with the specific polypeptide employed; and like factorswell known in the medical arts. For example, it is well within the skillof the art to start doses of the compound at levels lower than thoserequired to achieve the desired therapeutic effect and to graduallyincrease the dosage until the desired effect is achieved. However, thedaily dosage of the products may be varied over a wide range from 0.01to 1,000 mg per adult per day. Preferably, the compositions contain0.01, 0.05, 0.1, 0.5, 1.0, 2.5, 5.0, 10.0, 15.0, 25.0, 50.0, 100, 250and 500 mg of the active ingredient for the symptomatic adjustment ofthe dosage to the patient to be treated. A medicament typically containsfrom about 0.01 mg to about 500 mg of the active ingredient, preferablyfrom 1 mg to about 100 mg of the active ingredient. An effective amountof the drug is ordinarily supplied at a dosage level from 0.0002 mg/kgto about 20 mg/kg of body weight per day, especially from about 0.001mg/kg to 7 mg/kg of body weight per day.

Screening Methods

A further aspect of the invention relates a method for screening an FLT3receptor antagonist for the treatment or prevention of pain disorders.

For example, the screening method may measure the binding of a candidatecompound to FLT3, or to cells or membranes bearing FLT3, or a fusionprotein thereof by means of a label directly or indirectly associatedwith the candidate compound.

Alternatively, a screening method may involve measuring or,qualitatively or quantitatively, detecting ability of said candidatecompound to modulate the intracellular [Ca²⁺], and efficiently treatsand protects against pain disorders.

In a particular embodiment, the screening method of the inventioncomprises the steps consisting of:

a) providing a plurality of cells expressing FLT3 on their surface;

b) incubating said cells with a candidate compound;

c) determining whether said candidate compound binds to and inhibitsFLT3; and

d) selecting the candidate compound that binds to and inhibits FLT3.

In a particular embodiment, the screening method of the invention mayfurther comprising a step consisting of administering the candidatecompound selected at step d) to an animal model of pains disorders tovalidate the therapeutic and/or protective effects of said candidatecompound on pains disorders.

In one preferred embodiment, the FLT3 receptor antagonist is a selectiveFLT3 receptor antagonist.

In general, such screening methods involve providing appropriate cellswhich express FLT3 on their surface. In particular, a nucleic acidencoding FLT3 may be employed to transfect cells to thereby express thereceptor of the invention. Such a transfection may be accomplished bymethods well known in the art.

In a particular embodiment, said cells may be selected from the groupconsisting of the mammal cells reported yet to express FLT3 (e.g. dorsalroot ganglia (DRG) neurones).

The screening method of the invention may be employed for determining anantagonist by contacting such cells with compounds to be screened anddetermining whether such compound inhibits FLT3.

In another particular embodiment, the screening method of the inventioncomprises the steps consisting:

a) determining the ability of a candidate compound to inhibit theinteraction between a FLT3 polypeptide and a FL polypeptide and

b) selecting positively the candidate compound that inhibits saidinteraction.

At step a), any method suitable for the screening of protein-proteininteractions is suitable.

Whatever the embodiment of step a) of the screening method, the completeFLT3 protein and the complete FL protein may be used as the bindingpartners. It should further be noted that fragments of FLT3 protein andFL protein that include the site of interaction may be used as thebinding partners.

The term “polypeptide” means herein a polymer of amino acids having nospecific length. Thus, peptides, oligopeptides and proteins are includedin the definition of “polypeptide” and these terms are usedinterchangeably throughout the specification, as well as in the claims.The term “polypeptide” does not exclude post-translational modificationsthat include but are not limited to phosphorylation, acetylation,glycosylation and the like. Also encompassed by this definition of“polypeptide” are homologs thereof.

Accordingly, the term “FLT3 polypeptide” refers to the FLT3 protein or afragment thereof that comprises the site of interaction with FL protein.In the same manner, the term “FL polypeptide” refers to the FL proteinor a fragment thereof that comprises the site of interaction with FLT3protein.

The compounds that inhibit the interaction between FLT3 and FL encompassthose compounds that bind either to the FLT3 or to FL, provided that thebinding of the said compounds of interest then prevent the interactionbetween FLT3 and FL.

Polypeptides of the invention may be produced by any technique known perse in the art, such as without limitation, any chemical, biological,genetic or enzymatic technique, either alone or in combination(s).

Knowing the amino acid sequence of the desired sequence, one skilled inthe art can readily produce said polypeptides, by standard techniquesfor production of polypeptides. For instance, they can be synthesizedusing well-known solid phase method, preferably using a commerciallyavailable peptide synthesis apparatus (such as that made by AppliedBiosystems, Foster City, Calif.) and following the manufacturer'sinstructions.

Alternatively, the polypeptides of the invention can be synthesized byrecombinant DNA techniques as is now well-known in the art. For example,these fragments can be obtained as DNA expression products afterincorporation of DNA sequences encoding the desired (poly)peptide intoexpression vectors and introduction of such vectors into suitableeukaryotic or prokaryotic hosts that will express the desiredpolypeptide, from which they can be later isolated using well-knowntechniques.

Labelled Polypeptides

In one embodiment, the FLT3 polypeptide or the FL polypeptide of theinvention is labelled with a detectable molecule for the screeningpurposes.

Accordingly, said detectable molecule may consist of any compound orsubstance that is detectable by spectroscopic, photochemical,biochemical, immunochemical or chemical means. For example, usefuldetectable molecules include radioactive substance (including thosecomprising 32P, 25S, 3H, or 125I), fluorescent dyes (including5-bromodesosyrudin, fluorescein, acetylaminofluorene or digoxigenin),fluorescent proteins (including GFPs and YFPs), or detectable proteinsor peptides (including biotin, polyhistidine tails or other antigen tagslike the HA antigen, the FLAG antigen, the c-myc antigen and the DNPantigen).

According to the invention, the detectable molecule is located at, orbound to, an amino acid residue located outside the said amino acidsequence of interest, in order to minimise or prevent any artefact forthe binding between said polypeptides or between the candidate compoundand or any of said polypeptides.

Fluorescence Assays

In another embodiment of the screening method of the invention, the FLT3polypeptide and the FL polypeptide as above defined are labelled with afluorescent molecule or substrate. Therefore, the potential inhibitioneffect of the candidate compound to be tested on the binding between theFLT3 polypeptide and the FL polypeptide as above defined is determinedby fluorescence quantification.

For example, the FLT3 polypeptide and the FL polypeptide as abovedefined may be fused with auto-fluorescent polypeptides, as GFP or YFPsas above described. The FLT3 polypeptide and the FL polypeptide as abovedefined may also be labelled with fluorescent molecules that aresuitable for performing fluorescence detection and/or quantification forthe binding between said polypeptides using fluorescence energy transfer(FRET) assay. The FLT3 polypeptide and the FL polypeptide as abovedefined may be directly labelled with fluorescent molecules, by covalentchemical linkage with the fluorescent molecule as GFP or YFP. The FLT3polypeptide and the FL polypeptide as above defined may also beindirectly labelled with fluorescent molecules, for example, by noncovalent linkage between said polypeptides and said fluorescentmolecule. Actually, said FLT3 polypeptide and FL polypeptide as abovedefined may be fused with a receptor or ligand and said fluorescentmolecule may be fused with the corresponding ligand or receptor, so thatthe fluorescent molecule can non-covalently bind to said FLT3polypeptide and FL polypeptide. A suitable receptor/ligand couple may bethe biotin/streptavidin paired member or may be selected among anantigen/antibody paired member. For example, a polypeptide according tothe invention may be fused to a poly-histidine tail and the fluorescentmolecule may be fused with an antibody directed against thepoly-histidine tail.

As already specified, step a) of the screening method of the inventionencompasses determination of the ability of the candidate compound toinhibit the interaction between the FLT3 polypeptide and the FLpolypeptide as above defined by fluorescence assays using FRET. Thus, ina particular embodiment, the FLT3 polypeptide as above defined islabelled with a first fluorophore substance and the FL polypeptide islabelled with a second fluorophore substance. The first fluorophoresubstance may have a wavelength value that is substantially equal to theexcitation wavelength value of the second fluorophore, whereby the bindof said first and second polypeptides is detected by measuring thefluorescence signal intensity emitted at the emission wavelength of thesecond fluorophore substance. Alternatively, the second fluorophoresubstance may also have an emission wavelength value of the firstfluorophore, whereby the binding of said and second polypeptides isdetected by measuring the fluorescence signal intensity emitted at thewavelength of the first fluorophore substance.

The fluorophores used may be of various suitable kinds, such as thewell-known lanthanide chelates. These chelates have been described ashaving chemical stability, long-lived fluorescence (greater than 0.1 mslifetime) after bioconjugation and significant energy-transfer inspecificity bioaffinity assay.

In a preferred embodiment, the fluorescence assay performed at step a)of the screening method consists of a Homogeneous Time ResolvedFluorescence (HTRF) assay, such as described in document WO 00/01663 orU.S. Pat. No. 6,740,756, the entire content of both documents beingherein incorporated by reference. HTRF is a TR-FRET based technologythat uses the principles of both TRF (time-resolved fluorescence) andFRET. More specifically, the one skilled in the are may use a HTRF assaybased on the time-resolved amplified cryptate emission (TRACE)technology as described in Leblanc et al., 2002. The HTRF donorfluorophore is Europium Cryptate, which has the long-lived emissions oflanthanides coupled with the stability of cryptate encapsulation. XL665,a modified allophycocyanin purified from red algae, is the HTRF primaryacceptor fluorophore. When these two fluorophores are brought togetherby a biomolecular interaction, a portion of the energy captured by theCryptate during excitation is released through fluorescence emission at620 nm, while the remaining energy is transferred to XL665. This energyis then released by XL665 as specific fluorescence at 665 nm. Light at665 nm is emitted only through FRET with Europium. Because EuropiumCryptate is always present in the assay, light at 620 nm is detectedeven when the biomolecular interaction does not bring XL665 within closeproximity.

Therefore in one embodiment, step a) of the screening method maytherefore comprise the steps consisting of:

(1) bringing into contact a pre-assay sample comprising:

-   -   a FLT3 polypeptide fused to a first antigen,    -   a FL polypeptide fused to a second antigen    -   a candidate compound to be tested

(2) adding to the said pre assay sample of step (2):

-   -   at least one antibody labelled with a European Cryptate which is        specifically directed against the first said antigen    -   at least one antibody labelled with XL665 directed against the        second said antigen

(3) illuminating the assay sample of step (2) at the excitationwavelength of the said European Cryptate

(4) detecting and/or quantifying the fluorescence signal emitted at theXL665 emission wavelength

(5) comparing the fluorescence signal obtained at step (4) to thefluorescence obtained wherein pre assay sample of step (1) is preparedin the absence of the candidate compound to be tested.

If at step (5) as above described, the intensity value of thefluorescence signal is different (lower or higher) than the intensityvalue of the fluorescence signal found when pre assay sample of step (1)is prepared in the absence of the candidate compound to be tested, thenthe candidate compound may be positively selected at step b) of thescreening method.

Antibodies labelled with a European Cryptate or labelled with XL665 canbe directed against different antigens of interest including GST,poly-histidine tail, DNP, c-myx, HA antigen and FLAG which include. Suchantibodies encompass those which are commercially available from CisBio(Bedfors, Mass., USA), and notably those referred to as 61GSTKLA or61HISKLB respectively.

According to one embodiment of the invention, the candidate compoundsmay be selected from a library of compounds previously synthesised, or alibrary of compounds for which the structure is determined in adatabase, or from a library of compounds that have been synthesised denovo or natural compounds.

The candidate compound may be selected from the group of (a) proteins orpeptides, (b) nucleic acids and (c) organic or chemical compounds(natural or not).

Such the method may be used to screen FLT3 receptor antagonist accordingto the invention.

Pharmaceutical Compositions

The FLT3 receptor antagonist or inhibitor of FLT3 receptor geneexpression may be combined with pharmaceutically acceptable excipients,and optionally sustained-release matrices, such as biodegradablepolymers, to form therapeutic compositions.

In the pharmaceutical compositions of the present invention, the activeprinciple, alone or in combination with another active principle, can beadministered in a unit administration form, as a mixture withconventional pharmaceutical supports, to animals and human beings.Suitable unit administration forms comprise oral-route forms such astablets, gel capsules, powders, granules and oral suspensions orsolutions, sublingual and buccal administration forms, aerosols,implants, subcutaneous, transdermal, topical, intraperitoneal,intramuscular, intravenous, subdermal, transdermal, intrathecal andintranasal administration forms and rectal administration forms.

Preferably, the pharmaceutical compositions contain vehicles which arepharmaceutically acceptable for a formulation capable of being injected.These may be in particular isotonic, sterile, saline solutions(monosodium or disodium phosphate, sodium, potassium, calcium ormagnesium chloride and the like or mixtures of such salts), or dry,especially freeze-dried compositions which upon addition, depending onthe case, of sterilized water or physiological saline, permit theconstitution of injectable solutions.

The pharmaceutical forms suitable for injectable use include sterileaqueous solutions or dispersions; formulations including sesame oil,peanut oil or aqueous propylene glycol; and sterile powders for theextemporaneous preparation of sterile injectable solutions ordispersions. In all cases, the form must be sterile and must be fluid tothe extent that easy syringability exists. It must be stable under theconditions of manufacture and storage and must be preserved against thecontaminating action of microorganisms, such as bacteria and fungi.

Solutions comprising compounds of the invention as free base orpharmacologically acceptable salts can be prepared in water suitablymixed with a surfactant, such as hydroxypropylcellulose. Dispersions canalso be prepared in glycerol, liquid polyethylene glycols, and mixturesthereof and in oils. Under ordinary conditions of storage and use, thesepreparations contain a preservative to prevent the growth ofmicroorganisms.

The FLT3 receptor antagonist or inhibitor of FLT3 receptor geneexpression of the invention can be formulated into a composition in aneutral or salt form. Pharmaceutically acceptable salts include the acidaddition salts (formed with the free amino groups of the protein) andwhich are formed with inorganic acids such as, for example, hydrochloricor phosphoric acids, or such organic acids as acetic, oxalic, tartaric,mandelic, and the like. Salts formed with the free carboxyl groups canalso be derived from inorganic bases such as, for example, sodium,potassium, ammonium, calcium, or ferric hydroxides, and such organicbases as isopropylamine, trimethylamine, histidine, procaine and thelike.

The carrier can also be a solvent or dispersion medium containing, forexample, water, ethanol, polyol (for example, glycerol, propyleneglycol, and liquid polyethylene glycol, and the like), suitable mixturesthereof, and vegetables oils. The proper fluidity can be maintained, forexample, by the use of a coating, such as lecithin, by the maintenanceof the required particle size in the case of dispersion and by the useof surfactants. The prevention of the action of microorganisms can bebrought about by various antibacterial and antifungal agents, forexample, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, andthe like. In many cases, it will be preferable to include isotonicagents, for example, sugars or sodium chloride. Prolonged absorption ofthe injectable compositions can be brought about by the use in thecompositions of agents delaying absorption, for example, aluminiummonostearate and gelatin.

Sterile injectable solutions are prepared by incorporating the activepolypeptides in the required amount in the appropriate solvent withvarious of the other ingredients enumerated above, as required, followedby filtered sterilization. Generally, dispersions are prepared byincorporating the various sterilized active ingredients into a sterilevehicle which contains the basic dispersion medium and the requiredother ingredients from those enumerated above. In the case of sterilepowders for the preparation of sterile injectable solutions, thepreferred methods of preparation are vacuum-drying and freeze-dryingtechniques which yield a powder of the active ingredient plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof.

Upon formulation, solutions will be administered in a manner compatiblewith the dosage formulation and in such amount as is therapeuticallyeffective. The formulations are easily administered in a variety ofdosage forms, such as the type of injectable solutions described above,but drug release capsules and the like can also be employed.

For parenteral administration in an aqueous solution, for example, thesolution should be suitably buffered if necessary and the liquid diluentfirst rendered isotonic with sufficient saline or glucose. Theseparticular aqueous solutions are especially suitable for intravenous,intramuscular, subcutaneous and intraperitoneal administration. In thisconnection, sterile aqueous media which can be employed will be known tothose of skill in the art in light of the present disclosure. Forexample, one dosage could be dissolved in 1 ml of isotonic NaCl solutionand either added to 1000 ml of hypodermoclysis fluid or injected at theproposed site of infusion. Some variation in dosage will necessarilyoccur depending on the condition of the subject being treated. Theperson responsible for administration will, in any event, determine theappropriate dose for the individual subject.

The FLT3 receptor antagonist or inhibitor of FLT3 receptor geneexpression of the invention may be formulated within a therapeuticmixture to comprise about 0.0001 to 1.0 milligrams, or about 0.001 to0.1 milligrams, or about 0.1 to 1.0 or even about 10 milligrams per doseor so. Multiple doses can also be administered.

In addition to the compounds of the invention formulated for parenteraladministration, such as intravenous or intramuscular injection, otherpharmaceutically acceptable forms include, e.g. tablets or other solidsfor oral administration; liposomal formulations; time release capsules;and any other form currently used.

The FLT3 receptor antagonist or inhibitor of FLT3 receptor geneexpression of the invention may also be used in combination with othertherapeutically active agents, for instance, inhibitors of receptortyrosine kinase (RTK) class III or RTK class VII inhibitors.

RTK class III (Platelet-derived Growth factor (PDGF) receptor family) isa class of receptor tyrosine kinases including c-KIT and c-fms.

RTK class VII (Tropomyosin-receptor-kinase (Trk) receptor family) is aclass of receptor tyrosine kinases including TrkA, TrkB and TrkC.

The foregoing therapeutically active agents are listed by way of exampleand are not meant to be limiting. Other therapeutically active agentswhich are currently available or that may be developed in the future areequally applicable.

Accordingly, pharmaceutical compositions of the invention as describedabove may further comprise one or more therapeutically active agentsselected in the group consisting of class III RTK inhibitors or classVII RTK inhibitors.

Moreover, if they are contained in different pharmaceuticalcompositions, said compositions may be administered to the patient atthe same time or successively. Thus, the present invention also relatesto a kit for the prevention or treatment of an inflammatory diseasecomprising a first pharmaceutical composition comprising a polypeptideor a compound according to the invention and a second pharmaceuticalcomposition comprising one or more therapeutically active agentsselected from the group consisting of class HI RTK inhibitors or classVII RTK inhibitors.

The invention will be further illustrated by the following figures andexamples. However, these examples and figures should not be interpretedin any way as limiting the scope of the present invention.

FIGURES

FIG. 1: FL induces thermal and mechanical hyperalgesia in vivo: FL orNGF (as positive control) was injected into mice hindpaws. (A)Behavioral analysis of the effect of FL injection on paw withdrawallatencies in response to radiant heat. **P<0.01. (B) Behavioral analysisof the effect of FL injection on the foot withdrawal response to vonFrey hair filaments. ***P<0.001.

FIG. 2: siRNA against FLT3 inhibits FL-induced thermal and mechanicalhyperalgesia in vivo: (A) Behavioral analysis of the effect ofintrathecally-delivered FLT3 siRNA on radiant heat induced pawwithdrawal latencies after FL injection. ***P<0.001. (B) Behavioralanalysis of the effect of intrathecally-delivered FLT3 siRNA on pawwithdrawal latencies in response to von Frey hair filaments after FLinjection. ***P<0.001.

FIG. 3: FL potentiates capsaicin activation of TRPV1 receptors inprimary sensory neurons in vitro: (A) ΔFpeak of capsaicin responsesbefore and after FL application in adult DRG neurons. ***P<0.001. (B)ΔFarea of capsaicin responses before and after FL application in adultDRG neurons. ***P<0.001.

FIG. 4: siRNA against FLT3 inhibits FL-induced potentiation ofcapsaicin-activated TRPV1 receptors in primary sensory neurons in vitro.(A) ΔFpeak of capsaicin responses in the absence or the presence of FLalone, FL with control siRNA, FL with FLT3 siRNA **P<0.01. (B) ΔFarea ofcapsaicin responses in the absence or the presence of FL alone, FL withcontrol siRNA, FL with FLT3 siRNA **P<0.01.

FIG. 5: The TRPV1 inhibitor sb-366791 inhibit FL-induced mechanicalhyperalgesia in vivo: Behavioral analysis of the effect ofintraplantar-delivered TRPV1 inhibitor sb-366791 (10 μl at 30 μg/ml) onthe foot withdrawal response to von Frey hair filaments after FLinjection. **P<0.01; ***P<0.001.

FIG. 6: CEP-701 and SU-11248 inhibits FL-induced potentiation ofcapsaicin-activated TRPV1 receptors in primary sensory neurons in vitro.ΔFpeak of capsaicin responses in the absence or the presence of FLalone, FL with CEP-701 (CEP; 200 nM), FL with SU-11248 (SUN; 25 nM)***P<0.001.

FIG. 7: BDT001 inhibits FL-binding to FLT3. Red-FL and increasingconcentrations of BDT001 were incubated with SNAP-FLT3-transfectedHEK293T cells and the binding of Red-FL was measured by time-resolvedfluorescence resonance energy transfer (TR-FRET). BDT-001 inhibited FLbinding in a concentration dependent manner with an of 17.2 μM. (Example2.1)

FIG. 8: BDT001 inhibited FL-induced mechanical hypersensitivity in mice.BDT001 (5 mg/kg) or vehicle (Veh) administered intraperitonallyinhibited mechanical pain hypersensitivity induced by FL, at 4 h, 24 hand 48 h post-administration. Bsl, basal scores before surgery. *P<0.05vs. Veh. By two-way ANOVA ANOVA for repeated measures and Bonferroni'spost-hoc test. (Example 2.2)

FIG. 9: BDT001 inhibits mechanical hypersensitivity induce dby nervelesion in the Chronic Constriction Injury model in mice. BDT001 (5 mg/kgi.p.) inhibited mechanical hypersensitivity in CCI-mice. After two basalscorings (Bsl), CCI or Sham was performed and mechanicalhypersensitivity measured 10 days later (CCI). Eleven days after sham orCCI, vehicle (Veh) or BDT001 (5 mg/kg i.p.) or vehicle (Veh) wasinjected and mechanical hypersensitivity measured 4, 24, 48 or 72 hafter injection. *P<0.05; ***P<0.001 vs veh by two-way ANOVA forrepeated measures and Bonferroni's post-hoc test. (Example 2.3)

EXAMPLE 1

Material & Methods

Animals:

Adult Swiss mice (8-10 week old) (CERJ, Le Genest St Isle, France) wereused according to the guidelines of the International Association forthe Study of Pain. Mice were housed in cages with a 12/12 hr light/darkcycle and fed food and water ad libitum.

Behavioral Experiments:

Behavioral responsiveness of the mice was tested following one week ofhabituation to the testing environment and the observer.

Mechanical Withdrawal Thresholds:

The sensitivity to punctuate mechanical stimuli was assessed using thedynamic plantar aesthesiometer (Bioseb, France). Each mouse was placedin a Plexiglass chamber. Sixty minutes later, a mechanical stimulus (asmall-diameter blunt metallic filament) was applied to the plantarsurface with an increasing force and the paw withdrawal latencies weremeasured. For each point, mice were tested three times and the responsesfor each paw were averaged.

Thermo-Nociceptive Testing:

Nociceptive threshold to acute thermal stimulation was measured usingthe paw retrieval test (Bioseb, France). Focused light from 12.5 Wprojection bulb was applied to the middle of the plantar surface of thehind paw (3 mm diameter). The projection bulb was turned off as soon asthe mouse removed its paw, and a digital timer connected in seriesmeasured the paw withdrawal latency to an accuracy of 0.1 s. We used acut-off latency of 15 s to avoid the possibility of tissue damage. Foreach point, mice were tested three times and the responses for each pawwere averaged.

Gene Knock-Down Experiments:

Small Interfering RNAs (siRNAs):

Pooled nontargeting control siRNA or specific siRNA against FLT3 (flk2)used in this study were the on-target plus SMART pools from Dharmacon(Perbio Science, Brebières, France; Dharmacon Catalog # L-002000-00-0005targeting FLT3.

Preparation of the RNA Polymer Complex:

The method used for intrathecal delivery was adapted from one previouslyreported (Tan et al., 2005). For polyethylenimine (PEI) complexformation, 5 μg specific or nonspecific siRNAs were complexed with 1.8μl of 200 μM linear low molecular weight PEI ExGen 500 (Euromedex,Souffelweyersheim, France). RNA-polymer complexes were allowed 10 min toform at room temperature. To allow visualization of transfected cells, 3mM dextran-tetramethylrhodamine (Invitrogen, Cergy Pontoise, France)were added to the 5% glucose solution containing the RNA-polymercomplex.

In Vivo Delivery of siRNA:

6-8 μl of the final solution were injected through the subarachnoidspace at the L5-S1 level of adult mice once a day for 5 days aspreviously described (Boudes et al, 2009). Animals were allowed 1 daysrecovery then were either used for behavioural testing or sacrificed andlumbar L4-L5 DRG collected and processed for [Ca2+]i imaging. Inpreliminary experiments, intrathecal transfection efficiency wasevaluated with a GFP siRNA tagged with rhodamine (Qiagen, Courtaboeuf,France) injected in an actin-GFP mice. Fluorescence analysis ofrhodamine tagged neurons on DRG slices demonstrated that 50-80% ofneurons were transfected with this method and that transfected neuronsdid not express GFP, evaluated as a lack of co-staining between greenGFP and red siRNA.

DRG Neurons Primary Culture:

Primary neuronal cultures were established from lumbar (L4 to L6) dorsalroot ganglia (DRG) of adult mice as previously described (Boudes et al,2009). Ganglia were treated twice with collagenase A (1 mg/ml, RocheDiagnostic, France) for 45 minutes (37° C.) and then with trypsin-EDTA(0.25%, Sigma, St Quentin Fallavier, France) for 30 minutes. They weremechanically dissociated by passing 8 to 10 times through the tip of afire-polished Pasteur pipette in neurobasal (Life Technologies, CergyPontoise, France) culture medium supplemented with 10% foetal bovineserum and DNase (50 U/ml, Sigma, St Quentin Fallavier, France). Isolatedcells were collected by centrifugation and suspended in neurobasalculture medium supplemented with 2% B27 (Life Technologies), 2 mMglutamine, penicillin/streptomycin (20 U/ml, 0.2 mg/ml). Dissociatedneurons were plated on poly-D,L-ornithine (0.5 mg/ml)-laminin (5μg/ml)-coated glass coverslips at a density of 2500 neurons per well andwere incubated in an incubator with a humidified 95% air/5% CO2atmosphere. Two hours after plating, the culture medium was carefullyremoved and replaced to eliminate dead cells and tissue debris. Thecells were maintained in culture at 37° C. until experiments wereperformed.

Measurements of Intracellular Ca²⁺ ([Ca²⁺]_(i)):

For intracellular Ca²⁺ measurements, DRG neurons were used 18-30 h afterplating as described previously. The cells were loaded with a Locksolution containing 2.5 μM Fura2-AM (Molecular probes, Invitrogen,France) by incubation at 37° C. for 30 minutes. The loading solution waswashed three times and the Fura2-AM was left to de-esterified for 20minutes at 37° C. During [Ca²⁺]_(i) measurements, perfusion rate of thecells was controlled with a gravity flow system, and the temperature wasmaintained at 37° C. using an in-line heating system (WarnerInstruments). Drugs were delivered with a rapid-switching localperfusion system.

Cells were imaged with a inverted microscope equipped with a NEOFLUAR25×0.8 objective lens (Axiovert 200, Zeiss, Le Pecq, France) and a CCDcamera (Cool SNAP ES, Roper Scientific, France). Lambda DG-4 filterchanger (Sutter Instrument, Novato, Calif., USA) was used for switchingbetween 340 nm and 380 nm excitation wavelengths. A Fura filter cubewith 400 long pass dichroic and D510/40m emission band pass was used tocollect fluorescence emissions separately for each wavelength. Imageswere acquired and analyzed with Metafluor software (Molecular Devices).Changes in intracellular calcium concentrations ([Ca²⁺]_(i)) weremonitored as changes of the ratio of the fura-2-fluorescence intensityrecorded at 340 nm and 380 nm excitation wavelengths (ΔF_(340/380)).

Cells with a robust response to high K+ application (50 mM) at the endof the protocol and a peak response to capsaicin (100 μM) >0.2ΔF_(340/380) were retained. Ca²⁺ peak response was easily distinguishedfrom optical noise (<0.02 ΔF_(340/380)). Capsaicin at 100 μM elicited aresponse >0.2 ΔF_(340/380) from the maximal number of cells and responsemagnitudes decreased with subsequent agonist presentation as previouslydescribed (Bonnington, J. K., and McNaughton, 2003).

Each neuron was stimulated three times by capsaicin at 4-5 min interval.Ca²⁺ response peak and area data are presented as the ratio of post-FLcapsaicin response to the second naïve capsaicin response in individualcells (ΔF_(peak), ΔF_(area)). Response areas were calculated as ameasure of total Ca²⁺ influx. The portion of the calcium response thatwas used for this measurement included the entire curve from theinitiation of the response until the point at which the calcium signalreturned to the prestimulus baseline. Typically, this occurred in <120s.

Unless otherwise stated, all standard chemicals were purchased fromSigma (France) except FL (from ABCYS SA, Paris, France). They weredissolved or conditioned in double distilled water or DMSO or in ethanolaccording to the recommendations suggested in the Merk Index-13thedition or recommendations from the suppliers. The osmolarity of all thesolutions ranged between 298 and 303 mosmol/l.

Statistics:

Results are presented as mean±S.E.M. For comparison between the twogroups, the data were analyzed by two-way ANOVA, and if warranted,followed t tests using the computer program Prism (GraphPad, San Diego,Calif.). A p value <0.05 was considered statistically significant.

Results

FL Produces Thermal and Mechanical Hyperalgesia In Vivo:

To investigate the potential role(s) of FL in modulating noxious heatsensitivity in vivo, we first injected FL (100 ng/10 μl) into micehindpaws, and latency to ipsilateral paw withdrawal from a radiant heatstimulus was measured. We observed a rapid and pronounced heathyperalgesia within one/two hours which return to control values after 6day. At 12H after injection, FL induced a significant thermalhyperalgesia (latency drop around 30%) compared to control mice hindpawsinjected with vehicle (p<0.001) (FIG. 1A). For comparison, NGF (100ng/10 μl) induced a significant latency drop of 25% at 12H (FIG. 1A).

We next tested the role of FL in modulating noxious mechanicalsensitivity in vivo. Injection of FL (100 ng/10 μl) into mice hindpawsresulted in a mechanical hyperalgesia within one/two hours whichreturned to control values after 6 day. At 12H after injection, FLreduced significantly (p<0.001) the foot withdrawal response to von Freyhair filaments to 50% compared to control mice hindpaws injected withvehicle (FIG. 1B). For comparison, NGF (100 ng/10 μl) induced a footwithdrawal response decrease of 30% at 12H (P<0.001) (FIG. 1B).

To test if a specific interaction between FL and its cognate highaffinity FLT3 receptor regulates the thermal and mechanical hyperalgesiainduced by paw injection of FL, we designed RNA interference experimentsto inhibit FLT3 expression (see Materials and Methods). Mice weredirectly injected into the L4-L5 subarachnoid space during 4 days eitherwith control siRNA (siRNA control group) or siRNA against FLT3 (1 μg/μlof siRNA) (siRNA FLT3 group). After hindpaw injection of FL, siRNAcontrol mice showed thermal and mechanical hyperalgesia to a similarextent as that of non siRNA treated mice at 4 h, 12 h, 1, 2, 3, 5, 7 and9 days post-injection. In marked contrast, mice that received siRNAagainst FLT3 did not show hyperalgesia throughout the 9-d test period(FIGS. 2A and 2B for comparison at 12 h post-injection; p<0.001).

These data suggest that FL, via its specific interaction with FLT3,plays a critical role in modulating noxious thermal and mechanical painsensitivity in vivo.

FL Potentiates the TRPV1-Dependent Capsaicin Responses in Sensory DRGNeurons In Vitro:

Previous results have shown that the capsaicin TRPV1 receptor isnecessary for inflammatory thermal hyperalgesia mediated by NGF andthat, in vitro, NGF acutely potentiates TRPV1 functioning. The nextexperiments were designed to test the hypothesis that FL modulates TRPV1signaling. We used FURA-2-based calcium imaging to quantify the increasein [Ca2+]i that follows capsaicin application to TRPV1 positive sensoryneurons. As previously shown, repetitive capsaicin puffs induced TRPV1channel responses that showed significant tachyphylaxis which reflects adesensitization of the ionotropic receptor (Bonnington, J. K., andMcNaughton, 2003). When the cells are exposed to NGF between theapplications, a maintained or an increased TRPV1 response magnitude isobserved, indicating a sensitization of TRPV1 receptor by NGF(Bonnington, J. K., and McNaughton, 2003). In the present study,potentiated cells were defined as having a response to capsaicin greaterin magnitude to the mean of the control response plus two standarddeviations. At 10 and 100 ng/ml, FL induced a very robust potentiationof the capsaicin response in ≈56% of the cells that respond to capsaicin(FIG. 3A). In our standard protocol, FL was concomitantly applied withthe third capsaicin application indicating that the effect of FL may bevery rapid. The mean fold increase in TRPV1 response (that include allcapsaicin responders, both potentiated and unaffected, in thepopulation) was ≈212% for ΔFpeak and ≈512% for ΔFarea (p<0.001) (FIG.3B).

Here again, we designed RNA interference experiments to inhibit FLT3expression in order to test if a specific interaction between FL and itscognate high affinity FLT3 receptor regulated the potentiation ofcapsaicin-evoked responses in sensory neurons by FL (see Materials andMethods). Capsaicin-responder sensory neurons injected with siRNAcontrol showed a FL-induced sensitization in [Ca2+]i response similar tonon-transfected control neurons (FIGS. 4 A and B). In marked contrast,neurons that received siRNA against FLT3 did not show any potentiationof the capsaicin response after FL application in comparison to controland control siRNA-treated sensory neurons (p<0.001) (FIGS. 4 A and B).

To investigate the potential role(s) of FL in modulating noxioussensitivity through the activation of TRPV1 receptor in vivo, we testedthe effect of the TRPV1 inhibitor sb-366791 on the FL-induced mechanicalhyperalgesia. At 12 h after injection, FL reduced significantly the footwithdrawal response latency to von Frey hair filaments compared tocontrol mice hindpaws injected with vehicle as described above (FIG. 2A;FIG. 5). In marked contrast, mice that received sb-366791 (intraplantarinjection of 10 μl at 30 μg/ml, one hour before FL injection), did notshow any change in the foot withdrawal response to von Frey hairfilaments in comparison with control mice (FIG. 5).

The present data strongly suggest that FL potentiates in vitro thefunctioning of the capsaicin-sensitive TRPV1 ionotropic receptor in asubpopulation of nociceptive sensory neurons.

Kinase Inhibitors with Activity Against FLT3 Inhibits FL-InducedPotentiation of Capsaicin-Activated TRPV1 Receptors in Primary SensoryNeurons In Vitro:

Several small molecules kinase inhibitor with activity against FLT3,both in vitro and in vivo, are already available. Two of them, alreadyused in clinical trials, CEP-701 (lestaurtinib) and sunitinib(SU-11248), were tested for their capacity to inhibit the potentiationof capsaicin-evoked responses in sensory neurons by FL in vitro.

Neurons incubated in the presence of either CEP-701 (200 nM) orsunitinib (25 nM) did not show any sensitization in capsaicin [Ca2+]iresponse after FL application in contrast to control sensory neurons(p<0.001) (FIG. 6).

Altogether, these finding are consistent with the idea that treatmentwith FL, through a specific interaction with its high-affinity cognatereceptor FLT3, can produce acute thermal and mechanical hyperalgesia invivo, probably by directly sensitizing TRPV1 receptor in a subpopulationof nociceptors.

EXAMPLE 2

This example describes BDT001 as a small organic molecule(N-(5-chloro-2-hydroxyphenyl)-3-(piperidin-1-ylsuflonyl)benzamide),which is an FLT3 receptor antagonist that is an inhibitor of theinteraction between FLT3 and FL, according to one embodiment of thepresent invention. BDT001 is efficacious to antagonize FL-induced painand treat pain in an animal model of neuropathic pain. Said molecule waspreviously disclosed in WO2016/016370 as example 1 (page 38-39)(thecomplete contents of which are herein incorporated by reference). Itssynthesis is in particular described in said document.

2.1. BDT001 Inhibits FL Binding to FLT3.

A recombinant rh-FLT3-L was produced as previously reported (Verstraeteet al., 2009) and labeled with a red fluorescent probe (Red-FL) byCisbio Bioassays (Codolet, France), which also provided a plasmidencoding for the SNAP tagged human FLT3 (pSNAP-FLT3) used for transienttransfection of HEK293T cells. Transfections were performed in 96 wellplates using cell density of 50,000 cells per well. Prior to cellplating, wells were pre-coated with Poly-L-Ornithine (50 μl of 10 mg/ml)for 30 minutes at 37° C. Transfection mixes were prepared using 100 ngof SNAP-FLT3 plasmid, 0.25 μl of lipofectamine 2000 (Invitrogen) and 50μl Opti-MEM per well. Prior to their addition in plates, transfectionmixes were preincubated for 20 min at room temperature. Then 100 μl ofHEK293T cells, maintained in DMEM Glutamax (Invitrogen) supplementedwith antibiotics (penicillin 50 U/ml, streptomycin 50 μg/ml) and 10%heat-inactivated Foetal Calf were plated at a density of 500,000cells/ml in each well and were incubated at 37° C. under 5% CO2 for 24h.

For the FL binding assay, Red-FL and BDT001 were diluted in Tag-litelabelling medium (Cisbo Bioassays, ref. LABMED). SNAP-FLT3 transfectedcells were incubated with 0.5 nM Red-FL in the presence of increasingconcentrations of BDT001. In the plates containing labelled cells, 50 μl(or 10 μl in 384-well plates) of Tag-lite labelling medium, 25 μl (or 5μl in 384-well plates) of compounds to be tested were added andincubated for 1 h at room temperature prior to the addition of 25 μl (or5 μl in 384-well plates) of Red-FL. Plates were then incubated at roomtemperature for 4 h or overnight before signal detection. Thetime-resolved fluorescence resonance energy transfer (TR-FRET) signalwas detected using an advanced fluorescence microplate reader (RUBYstar,BMG Labtech) equipped with a HTRF optic module allowing a donorexcitation at 337 nm and a signal collection both at 665 nm and 620 nm.A frequency of 20 flashes/well is selected for the laser excitation. Thesignal was collected both at 665 nm and 620 nm using the followingtime-resolved settings: delay 50 μs, integration time 400 μs. HTRFratios were obtained by dividing the acceptor signal (665 nm) by thedonor signal (620 nm).

BDT001 was tested at concentrations ranging from 2·10⁻⁷M to 2·10⁻⁴ M andinhibited FL binding with an IC₅₀ of 17.2 μM (FIG. 7).

2.2. BDT001 Inhibits FL-Induced Mechanical Hypersensitivity In Vivo inMice.

After two basal score measurement, FL (50 ng/10 μl) was injectedintrathecally to mice and mechanical hypersensitivity) and FL-inducedmechanical withdrawal threshold was measured as in Example 1. BDT001 (5mg/kg) administered intraperitonally inhibited mechanical painhypersensitivity, induced by FL, at 24 h and 48 h post-administration(FIG. 8).

2.3. BDT001 Inhibits Nerve Injury-Induced Mechanical Hypersensitivity.

The Chronic Constriction Injury (CCI) model is well-recognized animalmodel of persistent peripheral neuropathic pain, consisting in threechronic ligatures tied loosely around the sciatic nerve (Bennett andXie, 1988). C57BL/6 mice (Janvier, France) were anesthetized and theskin was incised and the sciatic nerve was exposed unilaterally at themild-high level by dissecting through the biceps femoris. Threeligations (catgut 6.0) were loosely tied around the sciatic nerve withabout 1 mm spacing to reduce blood flow. The skin was then closed withstaples. In sham-operated animals the sciatic nerve was exposed withoutligation. For behavioral testing, a 0.6 g-von Frey filament was used formeasuring hindpaw mechanical hypersensitivity. Sharp withdrawal of thestimulated hindpaw was considered as a positive response. The procedurewas applied 10 times and the percentage of positive responses wascalculated.

As shown in FIG. 9, a large mechanical hypersensitivity was produced 10days after the CCI-surgery, as measured by the increase of withdrawalresponses. A single administration of BDT001 (5 mg/kg, intraperitonally)almost completely reversed mechanical hypersensitivity at 4 h, 24 h and48 h post-administration in CCI-operated mice, but not in sham-operatedmice.

The above results show that BDT001 is an FLT3 antagonist that inhibitsFL interaction with FLT3 and FL-induced mechanical hypersensitivity andcan be used to treat pain symptoms in an animal model of neuropathicpain.

REFERENCES

Throughout this application, various references describe the state ofthe art to which this invention pertains. The disclosures of thesereferences are hereby incorporated by reference into the presentdisclosure.

-   Bennett, G J & Xie, Y K. A peripheral mononeuropathy in rat that    produces disorders of pain sensation like those seen in man. Pain    33, 87-107 (1988). Bonnington, J. K., and McNaughton, P. A. (2003).    Signalling pathways involved in the sensitisation of mouse    nociceptive neurones by nerve growth factor. J. Physiol. 551,    433-446.-   Boudes, M, Sar, C, Menigoz, A, Hilaire, C, Pequignot, M, Kozlenkov,    V, Marmorstein, A, Carroll, P, Valmier, J, Scamps, F (2009). Best1/    is a gene regulated by nerve injury and required for Câ2+-activated    Cl̂− current expression in sensory neurons in mice. J. Neurosci. 29,    46, 10060-68-   Caterina, M. J., Leffler, A., Malmberg, A. B., Martin, W. J.,    Trafton, J., Petersen-Zeitz, K. R., Koltzenburg, M., Basbaum, A. I.,    and Julius, D.; Impaired nociception and pain sensation in mice    lacking the capsaicin receptor; Science 2000 288, 306-313.-   Chuang, H. H., Prescott, E. D., Kong, H., Shields, S., Jordt, S. E.,    Basbaum, A. I., Chao, M. V., and Julius, D.; Bradykinin and nerve    growth factor release the capsaicin receptor from    PtdIns(4,5)P2-mediated inhibition; Nature 2001 411, 957-962.-   Davis, J. B., Gray, J., Gunthorpe, M. J., Hatcher, J. P., Davey, P.    T., Overend, P., Harries, M. H., Latcham, J., Clapham, C., Atkinson,    K.; Vanilloid receptor-1 is essential for inflammatory thermal    hyperalgesia; Nature 2000 405, 183-187.-   Fabian M A, Biggs W H 3rd, Treiber D K, et al. A small    molecule-kinase interaction map for clinical kinase inhibitors. Nat    Biotechnol. 2005; 23(3):329-336.-   Jordt, S. E., McKemy, D. D., and Julius, D.; Lessons from peppers    and peppermint: the molecular logic of thermosensation; Curr Opin    Neurobiol 200313, 487-492.-   Karaman M W, Herrgard S, Treiber D K, et al. A quantitative analysis    of kinase inhibitor selectivity. Nat Biotechnol. 2008;    26(1):127-132.-   Li Y, Li H, Wang M N, Lu D, Bassi R, Wu Y, Zhang H, Balderes P,    Ludwig D L, Pytowski B, Kussie P, Piloto O, Small D, Bohlen P, Witte    L, Zhu Z, Hicklin D J. Suppression of leukemia expressing wild-type    or ITD-mutant FLT3 receptor by a fully human anti-FLT3 neutralizing    antibody; Blood. 2004 Aug. 15; 104(4):1137-44.-   Malin, S. A., Molliver, D. C., Koerber, H. R., Cornuet, P., Frye,    R., Albers, K. M., and Davis, B. M.; Glial cell line-derived    neurotrophic factor family members sensitize nociceptors in vitro    and produce thermal hyperalgesia in vivo; J Neurosci 2006 26,    8588-8599.-   Sternberg D W, Licht J D; Therapeutic intervention in leukemias that    express the activated fms-like tyrosine kinase 3 (FLT3):    opportunities and challenges; Curr Opin Hematol. 2005 January;    12(1):7-13.-   Verstraete K, Koch S, Ertugrul S, Vandenberghe I, Aerts M,    Vandriessche G, Thiede C, Savvides S N. Efficient production of    bioactive recombinant human Flt3 ligand in E. coli. Protein J 28,    57-65 (2009).-   Zarrinkar P P, Gunawardane R N, Cramer M D, Gardner M F, Brigham D,    Belli B, Karaman M W, Pratz K W, Pallares G, Chao Q, Sprankle K G,    Patel H K, Levis M, Armstrong R C, James J, Bhagwat S S; AC220 is a    uniquely potent and selective inhibitor of FLT3 for the treatment of    acute myeloid leukemia (AML); Blood. 2009 Oct. 1; 114(14):2984-92.-   Verstraete K, et al. Efficient production of bioactive recombinant    human Flt3 ligand in E. coli. Protein J 28, 57-65 (2009).

1. A method of treating or preventing pain disorders in a patient inneed thereof, comprising the step of administering to said patient atherapeutic amount of an FLT3 receptor antagonist, wherein said FLT3receptor antagonist is an inhibitor of the interaction between FLT3 andFL.
 2. The method of claim 1, wherein said pain disorders are selectedin the group consisting of acute pain, chronic pain, neuropathic pain,inflammatory pain, low back pain, post-operative pain, cancer pain,vascular headache such as migraine and hyperalgesia such as mechanicaland thermal hyperalgesia, allodynia such as thermal and mechanicalallodynia, peripheral sensitization of pain mechanisms and centralsensitization of pain mechanisms.
 3. A pharmaceutical composition forthe treatment or the prevention of pain which comprises a FLT3 receptorantagonist as defined in claim
 1. 4. A method for screening an FLT3receptor antagonist for the treatment or the prevention of paindisorders comprising the steps consisting of: a) determining the abilityof a candidate compound to inhibit the interaction between a FLT3polypeptide and a FL polypeptide, and b) selecting positively thecandidate compound that inhibits said interaction.